title: Detection and species identification of malaria parasites by isothermal tHDA amplification directly from human blood without sample preparation.
abstract: We report the clinical and analytical performance of an isothermal thermophilic helicase-dependent amplification assay for blood Plasmodium parasite detection and species-level identification. The assay amplifies the 18S rRNA gene fragment of all Plasmodium species and uses a species-specific probe and a pan-malarial probe to definitively identify Plasmodium falciparum from other infectious Plasmodium species. Amplicon-probe hybridization products are detected with a disposable dipstick enclosed in a cassette. With a pan-malarial-positive and P. falciparum-negative result, an additional test is performed to detect if the pan-malarial-positive band was the result of the presence of Plasmodium vivax. The assay uses only 2 μL of human whole blood directly for a 50-μL amplification reaction, without any pre-amplification processing. The clinical performance of the assay was validated using 88 samples from New York patients suspected of malaria or babesiosis. The overall sensitivity of the assay was 96.6% (95% CI, 87.3% to 99.4%), and the specificity was 100% (95% CI, 85.4% to 100%), compared with gold standard microscopy and a laboratory-developed molecular assay, respectively. The analytical sensitivity was 50 copies of DNA per assay or 200 parasites per microliter of blood, and the assay can detect samples with parasitemia levels <1%. This novel molecular diagnostic assay requires minimal laboratory instrumentation and uses un-processed blood as input; it can be readily performed in the field.
authors.affiliation: BioHelix Corporation, Beverly, Massachusetts, USA. li@biohelix.com
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.forename: Nirbhay
authors.initials: N
authors.lastname: Kumar
authors.forename: Anusha
authors.initials: A
authors.lastname: Gopalakrishnan
authors.forename: Christine
authors.initials: C
authors.lastname: Ginocchio
authors.forename: Ryhana
authors.initials: R
authors.lastname: Manji
authors.forename: Maureen
authors.initials: M
authors.lastname: Bythrow
authors.forename: Bertrand
authors.initials: B
authors.lastname: Lemieux
authors.forename: Huimin
authors.initials: H
authors.lastname: Kong
mesh_terms: D006801:Humans; D008288:Malaria / Q000175:diagnosis*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010961:Plasmodium / Q000145:classification* / Q000235:genetics*; D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S